Targeting of integrin-linked kinase with a small interfering RNA suppresses progression of experimental proliferative vitreoretinopathy.
Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts through its COOH terminus with beta1 and beta3 integrins, which mediates a diversity of cell functions by coupling integrins and growth factors to cascades of downstream signaling events. The purpose of this work was to investigate the effects of ILK on development of experimental proliferative vitreoretinopathy (PVR). Cultured human RPE cell line D407 was knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by MTT assay. Moreover, cell attachment, spreading, migration, microfilament dynamics, and cell cycling assays were performed. Furthermore, the impact of the ILK-specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of human RPE cells. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection. The results showed that blocking the expression of ILK by siRNA significantly inhibited human RPE cell attachment, spreading, migration and proliferation. The knockdown of ILK also disturbed F-actin assembly and induced a cellular arrest in the G1 phase of the cell cycle. Though the eyes injected with ILK-specific siRNA also developed features of PVR, the severities of day 28 post-injection were significantly lower than those in the control eyes (P<0.01). We conclude that targeting of ILK with a small interfering RNA not only inhibits human RPE cell attachment, spreading, migration and proliferation in vitro, but also effectively suppresses development of proliferative vitreoretinopathy in a rabbit model. This may be a potential therapeutic usefulness in treating PVR.
Guo
Lili
L
Department of Ophthalmology, People's Hospital, Peking University, Xizhimen South Street 11, Xi Cheng District, 100044 Beijing, China.
Yu
Wenzhen
W
Li
Xiaoxin
X
Zhao
Gang
G
Liang
Jianhong
J
He
Peiying
P
Wang
Kai
K
Zhou
Peng
P
Jiang
Yanrong
Y
Zhao
Mingwei
M
D008841:Actin Cytoskeleton / Q000502:physiology; D000818:Animals; D002448:Cell Adhesion; D016923:Cell Death; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D004195:Disease Models, Animal; D018450:Disease Progression; D015536:Down-Regulation; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011817:Rabbits; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000201:enzymology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection; D018630:Vitreoretinopathy, Proliferative / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D000099200:Scaffold Protein ILK
D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000099200:Scaffold Protein ILK